The present invention relates to recombinant DNA that encodes the MspA1I restriction endonuclease (MspA1I endonuclease or MspA1I) as well as MspA1I methyltransferase (MspA1I methylase or M.MspA1I), expression of MspA1I endonuclease and methylase in E. coli cells containing the recombinant DNA.
MspA1I endonuclease is found in the strain of Moraxella species (NEB#775, New England Biolabs"" strain collection). It recognizes the double-stranded DNA sequence 5xe2x80x2-CMG/CKG-3xe2x80x2 (M=A or C; K=G or T, / indicates the cleavage position) and cleaves between the G and C to generate blunt ends. MspA1I methylase (M.MspA1I) is also found in the same strain, which recognizes the same DNA sequence and presumably modifies the cytosine at the N4 position on hemi-methylated or non-methylated MspA1I sites.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases recognize and bind particular sequences of nucleotides (the xe2x80x98recognition sequencexe2x80x99) along the DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and twenty-eight restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 29:268-269 (2001)).
Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5xe2x80x2-TTT/AAA-3xe2x80x2, 5xe2x80x2-PuG/GNCCPy-3xe2x80x2 and 5xe2x80x2-CACNNN/GTG-3xe2x80x2 respectively. Escherichia coli RY13, on the other hand, produces only one Type II enzyme, EcoRI, which recognizes the sequence 5xe2x80x2-G/AATTC-3xe2x80x2.
A second component of bacterial/viral restriction-modification (R-M) systems are the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiably foreign DNA, is sensitive to restriction endonuclease recognition and cleavage. During and after DNA replication, usually the hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction digestion.
With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, i.e. populations of clones derived by xe2x80x98shotgunxe2x80x99 procedures, when they occur at frequencies as low as 10xe2x88x923 to 10xe2x88x924. Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719, (1980); HhaII: Mann et al., Gene 3:97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Since the expression of restriction-modification systems in bacteria enables them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12: 3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509, (1985); Msp45I: Wayne et al. Gene 202:83-88, (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421, (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119, (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241, (1983)).
A more recent method, the xe2x80x9cendo-blue methodxe2x80x9d, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535; Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response signals following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that some positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.
There are three major groups of DNA methyltransferases based on the position and the base that is modified (C5 cytosine methylases, N4 cytosine methylases, and N6 adenine methylases). N4 cytosine and N6 adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632, (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer DNA sites resistant to restriction digestion. For example, Dcm methylase modification of 5xe2x80x2-CCWGG-3xe2x80x2 (W=A or T) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpG methylase can modify the CG dinucleotide and make the NotI site (5xe2x80x2-GCGGCCGC-3xe2x80x2) refractory to NotI digestion (New England Biolabs"" catalog, 2000-01, page 220; Beverly, Mass.). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.
Type II methylase genes have been found in many sequenced microbial genomes (GenBank, REBASE(trademark) (New England Biolabs, Inc., Beverly, Mass.)). Direct PCR cloning and over-expression of ORFs adjacent to the methylase genes resulted in discovery of novel restriction enzyme specificities (Kong, et al., Nucl. Acid Res. 28:3216-3223 (2000)).
Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant molecules in the laboratory, there is a strong commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes and methylases. Such over-expression strains should also simplify the task of enzyme purification.
The present invention relates to a method for cloning MspA1I restriction gene (mspA1IR) from Moraxella species into E. coli by multiple inverse PCR and direct PCR from genomic DNA using primers that were based on the DNA sequences obtained via methylase selection.
It proved difficult to clone mspA1IR by screening a partial ApoI genomic DNA library. A second partial ApoI library was constructed using purified ApoI DNA fragments in the range of 3 to 10 kb. No positive methylase clones were ever identified. More complete genomic DNA libraries such as BamHI, EcoRI, HindIII, KpnI, SacI, SalI, SphI, and XbaI libraries were constructed and challenged with MspA1I endonuclease. Two resistant clones were identified with inserts encoding the MspA1I methylase in the BamHI library. The entire insert was sequenced. About 1900-bp sequence was derived upstream of the mspA1IM gene, but no apparent large ORFs were found that potentially encode the MspA1I endonuclease.
There is one truncated ORF of 281 bp downstream of the mspA1IM gene. This ORF did not show significant homology to any genes in GenBank. Therefore, efforts were made to obtain the sequence further downstream. After three rounds of inverse PCR reactions and direct sequencing of the PCR product, one ORF of 801 bp was found. This ORF, organized in the tail-to-tail orientation with the methylase gene, was the putative mspA1IR gene.
To over-express the putative mspA1IR gene and mspA1IM gene together in the same cell, both genes were amplified in PCR and cloned into expression vector pRRS. After screening 40 cell extracts of transformants, no over-expressing clones of MspA1I were detected. It was concluded that either the clones did not contain any insert, or did contain insert but expressed poorly due to under-methylation, or mutation(s) in the mspA1IR gene introduced in PCR. It was determined that a two-plasmid expression system may be more productive in over-expression of MspA1I.
Plasmid pUC19-MspA1IM was first introduced into T7 expression host ER2566 to fully modify the host chromosome. The mspA1IR gene was amplified by low cycles of PCR and inserted into a low-copy-number T7 expression vector pACYC-T7ter. The expression strain was ER2566 [pUC19-MspA1IM, pACYC-T7ter-MspA1IR]. After screening 25 cell extracts, 6 MspA1I over-expression clones were found. The mspA1IR insert of two expression clones were sequenced and confirmed to contain the same wild type sequence.